This invention relates to a method of increasing the specific activity of tissue plasminogen activator (tPA).
Tissue-type plasminogen activator (tPA) is an example of a glycoprotein whose biological properties are a reflection of the cooperative interaction of individual protein domains and the oligosaccharides at specific sites within the protein. The protein has a 527 amino acid sequence comprised of five domains: a fibronectin finger-like domain at the amino terminus, an epidermal growth factor-like domain, two kringle type domains and the serine protease domain at the carboxyl terminus [Pennica et al., Nature 301, 214-221 (1983); Banyai et al., Biochem. Biophys. Res. Commun. 125, 324-331 (1983)]. The serine protease domain catalyzes the conversion of plasminogen to plasmin, and this activity is stimulated by fibrin [Hoylaerts et al., J. Biol. Chem. 257, 2912-2919 (1982); Ranby, Biochem. Biophys. Acta 704, 461-469 (1982)]. Fibrin stimulation is thought to be mediated by binding sites for native fibrin in the fibronectin-like finger domain and for plasmin-degraded fibrin or lysine-like ligands in the second kringle domain [van Zonneveld et al., J. Biol. Chem. 261, 14214-14218 (1986); Verheijen et al., EMBO J. 5, 3525-3530 (1986)]. The function of the growth factor and the first kringle domains are less well established, but both are thought to be involved in the clearance of tPA from the circulation. Deletion of the growth factor domain prolongs the half-like of the tPA [Kalyan et al., J. Biol. Chem. 263, 3971-3978 (1988); Browne et al., Ibid. 263, 1599-1602 (1988)]. In the case of the first kringle domain, clearance appears to be mediated by the high mannose N-linked oligosaccharide side-chain present at Asn-117 within the kringle. Endo H treatment to selectively remove this oligosaccharide or site-directed mutagenesis to eliminate the glycosylation at this position results in a 3 to 5 fold longer circulating half-life [Lau et al., Bio/Technol. 5, 953-958 (1987); Hotchkiss et al., Thromb. Haem. 60, 255-261 (1988)]. In harmony with this, Kuiper et al., J. Biol. Chem 263, 18220-18224 (1988), found evidence for two separate clearance mechanisms. The liver parenchymal cell clearance of tPA was independent of carbohydrate, whereas tPA recognition by endothelial cells involved the mannose receptor.
In addition to this role in clearance, two observations suggest that both the enzymatic activity of tPA and its affinity for lysine are influenced by glycosylation. First, the type I glycoform (oligosaccharide at Asn-117, -184 and -448) and the type II glycoform (oligosaccharide at Asn-117 and -448 only) have been shown to have significantly different in vitro activities [Einarsson et al., Biochem. Biophys. Acta 830, 1-10 (1985); Wittwer et al., J. Cell Biol. 107, 584a (1988); Wittwer et al., Biochemistry 28, 7662-7669 (1989)]. Second, tPA isolated from different cell types have been shown to have different activities, even though the amino acid sequences are identical. Evidence was presented to support the hypothesis that the activity differences were the result of variations in oligosaccharide structure [Parekh et al., Biochemistry 28, 7644-7662 (1989); Howard et al., J. Cell. Biol. 107, 584a (1988); Wittwer et al., Biochemistry 28, 7662-7669 (1989)]. See also Feder et al., U.S. Pat. No. 4,751,084. Therefore, both the extent and type of oligosaccharide structures on the tPA protein appear to affect enzymatic activity.